Using Cell Cultures for the Investigation of Treatments for Attention Deficit Hyperactivity Disorder: A Systematic Review.

BACKGROUND: Advances in basic and molecular biology have promoted the use of cell cultures in a wide range of areas, including the evaluation of drug efficacy, safety and toxicity. OBJECTIVE: This article aims to provide a general overview of the methodological parameters of cell cultures used to investigate therapeutic options for Attention Deficit Hyperactivity Disorder. METHOD: A systematic search was performed in the electronic databases PubMed, Scopus, and DOAJ. In vitro experimental studies using cell cultures were included. RESULTS: A total of 328 studies were initially identified, with 16 included for qualitative synthesis. Seven studies used neuronal cells (SH-SY5Y neuroblastoma and PC12 cell line) and nine used nonneuronal cells. All the studies described the culture conditions, but most studies were inconsistent with regard to reporting results and raw data. Only one-third of the studies performed cell viability assays, while a further 30% conducted gene expression analysis. Other additional tests included electrophysiological evaluation and transporter activity. More than 50% of the studies evaluated the effects of drugs such as methylphenidate and atomoxetine, while plant extracts were assessed in four studies and polyunsaturated fatty acids in one. CONCLUSION: We suggested a flowchart to guide the planning and execution of studies, and a checklist to be completed by authors to allow the standardized reporting of results. This may guide the elaboration of laboratory protocols and further in vitro studies.

Proanthocyanidin Polymer-Rich Fraction of Stryphnodendron adstringens Promotes in Vitro and in Vivo Cancer Cell Death via Oxidative Stress.

Cervical cancer is the fourth most common cancer that affects women, mainly through human papilloma virus (HPV) infection with high-risk HPV16 and HPV18. The present study investigated the in vitro anticancer activity and mechanism of action of a proanthocyanidin polymer-rich fraction of Stryphnodendron adstringens (F2) in cervical cancer cell lines, including HeLa (HPV18-positive), SiHa (HPV16-positive), and C33A (HPV-negative) cells, and also evaluated in vivo anticancer activity. In vitro, cell viability was determined by the MTT assay. Cell migration was determined by the wound healing assay. The mechanism of action was investigated by performing ultrastructural analysis and evaluating reactive oxygen species (ROS) production, mitochondrial metabolism, lipoperoxidation, BCL-2 family expression, caspase expression, and DNA and cell membrane integrity. In vivo activity was evaluated using the murine Ehrlich solid tumor model. F2 time- and dose-dependently reduced cell viability and significantly inhibited the migration of cervical cancer cells. HeLa and SiHa cells treated with F2 (IC50) exhibited intense oxidative stress (i.e., increase in ROS and decrease in antioxidant species) and mitochondrial damage (i.e., mitochondrial membrane potential depolarization and a reduction of intracellular levels of adenosine triphosphate). Increases in the Bax/BCL-2 ratio and caspase 9 and caspase 3 expression, were observed, with DNA damage that was sufficient to trigger mitochondria-dependent apoptosis. Cell membrane disruption was observed in C33A cells (IC50 and IC90) and HeLa and SiHa cells (IC90), indicating progress to late apoptosis/necrosis. The inhibition of ROS production by N-acetylcysteine significantly suppressed oxidative stress in all three cell lines. In vivo, F2 significantly reduced tumor volume and weight of the Ehrlich solid tumor, and significantly increased lipoperoxidation, indicating that F2 also induces oxidative stress in the in vivo model. These findings indicate that the proanthocyanidin polymer-rich fraction of S. adstringens may be a potential chemotherapeutic candidate for cancer treatment.

The Effect of Phenazine-1-Carboxylic Acid on Mycelial Growth of Botrytis cinerea Produced by Pseudomonas aeruginosa LV Strain.

One of the most important postharvest plant pathogens that affect strawberries, grapes and tomatoes is Botrytis cinerea, known as gray mold. The fungus remains in latent form until spore germination conditions are good, making infection control difficult, causing great losses in the whole production chain. This study aimed to purify and identify phenazine-1-carboxylic acid (PCA) produced by the Pseudomonas aeruginosa LV strain and to determine its antifungal activity against B. cinerea. The compounds produced were extracted with dichloromethane and passed through a chromatographic process. The purity level of PCA was determined by reversed-phase high-performance liquid chromatography semi-preparative. The structure of PCA was confirmed by nuclear magnetic resonance and electrospray ionization mass spectrometry. Antifungal activity was determined by the dry paper disk and minimum inhibitory concentration (MIC) methods and identified by scanning electron microscopy and confocal microscopy. The results showed that PCA inhibited mycelial growth, where MIC was 25 µg mL-1. Microscopic analysis revealed a reduction in exopolysaccharide (EPS) formation, showing distorted and damaged hyphae of B. cinerea. The results suggested that PCA has a high potential in the control of B. cinerea and inhibition of EPS (important virulence factor). This natural compound is a potential alternative to postharvest control of gray mold disease.

Antifungal Activity of Condensed Tannins from Stryphnodendron adstringens: Effect on Candida tropicalis Growth and Adhesion Properties.

Candida species are some of the most common causes of fungal infection worldwide. The limited efficacy of clinically available antifungals warrants the search for new compounds for treating candidiasis. This study evaluated the effect of condensed tannin-rich fraction (F2 fraction) of Stryphnodendron adstringens on in vitro and in vivo growth of Candida tropicalis, and on yeast adhesion properties. F2 exhibited a fungistatic effect with the minimum inhibitory concentration ranging from 0.5 to 8.0 µg/mL. A significant reduction in biofilm mass was observed after either pretreatment of planktonic cells for 2 h (mean reduction of 46.31±8.17%) or incubation during biofilm formation (mean reduction of 28.44±13.38%) with 4x MIC of F2. Prior exposure of planktonic cells to this F2 concentration also significantly decreased yeast adherence on HEp-2 cells (mean reduction of 43.13±14.29%), cell surface hydrophobicity (mean reduction of 25.89±10.49%) and mRNA levels of the genes ALST1-3 (2.9-, 1.8- and 1.8-fold decrease, respectively). Tenebrio molitor larvae, which are susceptible to C. tropicalis infection, were used for in vivo testing. Treatment with 128 and 256 µg/mL F2 significantly increased the survival of infected larvae. These results indicate a combined effect of F2 on inhibition of yeast growth and interference in yeast adhesion, which may contribute to the suppression of infection caused by C. tropicalis, thus reinforcing the potential of the condensed tannins from S. adstringens for the development of novel antifungal agents.

Antiviral Activity of Trichilia catigua Bark Extracts for Herpesvirus and Poliovirus.

Herpesvirus and poliovirus are responsible for important diseases in human and animal. Trichilia catigua a Brazilian native plant known as catiguá has several medicinal properties among them antimicrobial for bacteria and protozoa, however, no antiviral activity has been reported yet. This study evaluated the antiviral activity of the crude extract (CE) and aqueous and ethyl acetate fractions (AF, EAF) obtained from T. catigua in the replication of the Herpes simplex virus (HSV-1), bovine herpesvirus (BoHV-1) and poliovirus (PV-1). The cytotoxicity was analyzed by MTT assay and the antiviral effect was determined by the addition of extracts (0.25 to 100.0 µg/ml), before (-2h and -1h), during (Oh) and after (1h and 2h) the viral infection, by plaque reduction assay, in HEp-2 cell culture. The virucidal activity and inhibition of viral adsorption were also evaluated. In addition, the combination index (CI) with Acyclovir (ACV - reference drug) was determined for HSV-1. CE, AF and EAF showed a low toxicity (CC(50) >400 µg/ml) and low inhibitory concentration (IC50), ranging from 2.44-34.25 µg/ml for herpesvirus and 0.67 to 1.8 µg/ml for PV-1, associated with high selectivity index. The tested compounds showed high virucidal effect and high ability to inhibit viral adsorption, for all virus. The CI demonstrated a synergic effect (CI<1) for AF and EAF comparatively to acyclovir (ACV). Our study demonstrated that the extract and fractions of T. catigua is promising for future antiviral drug design with economically feasible production.

Antimicrobial, cytotoxic and antioxidant activities and determination of the total tannin content of bark extracts Endopleura uchi.

Endopleura uchi is a typical Amazonian tree and its bark is popularly employed in the preparation of teas against myomas, arthritis, influenza, diarrhea and cancer. In this study, the antioxidant activity, cytotoxicity and antimicrobial activity of five different extracts of the bark, selected by their total tannin content, were assessed. The potential antioxidant activity of the extracts was determined by 2.2-diphenyl-1-picrylhydrazyl radical scavenging assay and the values found were very similar among the extracts and to the standards antioxidants used in the tests. Cytotoxicity analysis in mammalian cells indicated that all the tested extracts exhibited IC(50) values higher than the highest concentration used, showing that they do not present a risk when consumed under these conditions. Extract tested against five bacterial strains and one yeast strain did not show satisfactory growth inhibitory activity, and even the extracts that showed some antimicrobial activity were not effective at any dilution to determine the minimum inhibitory concentration. The results may serve as a reference for subsequent works, since such reference values described in the literature for the bark of E. uchi.

Antimicrobial and cytotoxic activities of medicinal plants of the Brazilian cerrado, using Brazilian cachaça as extractor liquid.

ETHNOPHARMACOLOGICAL IMPORTANCE: Many species of plants in the Brazilian cerrado (savanna) are widely used in ethnomedicine. However, the safety and effectiveness of medicinal plants used in communities with little or no access to manufactured drugs should be evaluated. AIM OF THE STUDY: Evaluate the antimicrobial and cytotoxic activities of extracts from eight plant species, obtained using Brazilian cachaça as the extractor liquid. MATERIALS AND METHODS: The extracts were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida parapsilosis, promastigote forms of Leishmania amazonensis, and poliovirus. In addition, cytotoxic activity was assayed in Vero cells and in human erythrocytes. RESULTS: The plant species Curatella americana, Sclerolobium aureum, and Plathymenia reticulata showed the best activity against yeasts, especially the crude extract of C. americana and its ethyl-acetate fraction. Kielmeyera lathrophyton showed a minimum inhibitory concentration of 250 µg/ml against S. aureus, and was inactive against gram-negative bacteria. The extract obtained from Annona coriacea showed the best activity against the promastigote forms of Leishmania amazonensis (IC(50)=175 µg/ml). Only C. americana showed potential for antipoliovirus activity. The concentrations of the crude extracts that showed toxicity to VERO cells had CC(50) between 31 and 470 µg/ml, and the lyophilized Brazilian cachaça showed a CC(50) of 307 µg/ml. None of the extracts showed toxicity against human erythrocytes. CONCLUSIONS: Among the plant species studied, C. americana proved to be effective against microorganisms, especially as an antifungal. The results will help in the search for alternative drugs to be used in pharmacotherapy, and will contribute to establish safe and effective use of phytomedicines in the treatment of infectious diseases.